Akkermansia muciniphila in infectious disease: A new target for this next-generation probiotic?

The common gastrointestinal commensal Akkermansia muciniphila is a mucin-degrading bacterium that is greatly reduced in individuals consuming a high-fat diet. Increasing evidence from a variety of clinical and pre-clinical studies suggests that oral supplementation with Akkermansia can improve metabolic health and moderate systemic inflammation. We and others have demonstrated a role for Akkermansia administration in protection against infectious disease and the outcome from sepsis. Very recent studies have indicated the molecular mechanisms by which A. muciniphila may interact with the host to influence systemic immune-regulation and control of microbial pathogenesis. Here we consider recent studies which demonstrate the efficacy of this potential next-generation probiotic in animal models of Salmonella Typhimurium, Listeria monocytogenes and Clostridioides difficile as well as influenza virus and phlebovirus. The potential mechanisms by which A. muciniphila may influence local and systemic immune responses are discussed.

capacity to directly influence cellular metabolism, such as the long-chain fatty acids palmitic and stearic acid, which engage the NLRP3 inflammasome to drive low-grade inflammation in the host. 1 Diet also has a significant influence on the gut microbiome, the composition of which impacts local inflammatory processes and gut barrier function through bacterial effectors such as short-chain fatty acids and bile acid modifications. 1 Perhaps unsurprisingly, in animal models, Western diets induce susceptibility to infection with foodborne pathogens including Listeria monocytogenes and Salmonella Typhimurium. 2,3In the Salmonella model, a high-fat (HF) diet increases bile salt production, providing the pathogen (which is bile tolerant) with a competitive advantage over the microbiota. 3Our laboratory has demonstrated a role for diet in L. monocytogenes infection in mice, with an HF diet reducing resistance to both local and systemic infection alongside concurrent changes to microbiome composition and immune function. 2In a subsequent study, we show that prior oral administration of Akkermansia muciniphila provides protection against infection with Listeria in the HF dietary model system with an apparent reversal of specific effects of an HF diet. 4 This commentary will contextualise those findings and point towards future work that is needed in this area if we are to understand and exploit the ability of the microbiota to protect against foodborne infection.
The role of the gut microbiota in resistance to Listeria infection has been demonstrated using germ-free animal models which generally demonstrate increased susceptibility to infection in comparison to conventionally raised animals. 5Mono-colonisation of gnotobiotic mice with Lactobacillus spp.prior to L. monocytogenes infection improved infection resistance, altered the expression pattern of interferon-stimulated genes in the host, and influenced the transcriptional response of the pathogen, suggesting potential mechanisms underpinning these findings. 6Antibiotic treatment of normal mice to reduce colonisation resistance prior to infection by L. monocytogenes significantly increases susceptibility to oral infection by the pathogen.Becattini et al. 7 utilised this model to identify individual commensal taxa that are important in resistance to infection, and which could potentially act as next-generation probiotics in the prevention of disease.
Similarly, diet may be used as a modifier of the microbiome to break colonisation resistance and allow the discovery of commensal strains that actively impede foodborne pathogens. 3It is well established that an HF Westernised diet causes a reduction in abundance of A. muciniphila. 1In our initial studies we showed that abundance of Akkermansia spp. was impacted even following short-term dietary changes to an HF diet and that this correlated with increased susceptibility to both oral and systemic L. monocytogenes infection. 2We then set up an experimental model in which mice were given HF diets either with or without the addition of supplemental live A. muciniphila and showed that Akkermansia significantly improved resistance to subsequent oral and systemic L. monocytogenes infection. 4We demonstrated that provision of Akkermansia did not significantly impact the composition of the microbiome.Similarly, even though Akkermansia is reported to facilitate the production of the short-chain fatty acid butyrate, which is associated with positive health effects in the gut, we did not detect differences in the microbial metabolic products in the caecum or faeces between groups. 4This suggests that the protective effects of Akkermansia are not the result of large-scale shifts in the microbiota but rather the direct action of Akkermansia itself on the local environment of the gut.
Locally, in our model, Akkermansia reduced goblet cell numbers in the colon of mice fed with an HF diet, 4 although this finding contrasts with another study which employed a longer period of HF dietary feeding. 8Goblet cells are a preferential site for cellular invasion by Listeria and this provides a potential local mechanism by which Akkermansia may enhance gastrointestinal, but not systemic, resistance to the pathogen. 9dditionally, Akkermansia may influence systemic infection resistance through a role in immune regulation, barrier function and homoeostasis.Indeed, a recent study showed that improved barrier function as a consequence of Akkermansia administration can protect against Clostridioides difficile infection in the colon. 10In our study, Akkermansia modified immune gene expression in the ileum (local) and liver (systemic), including regulation of genes encoding tumor necrosis factor-alpha (TNFα) both before and during Listeria infection. 4TNFα is well established as a key cytokine which enhances protection against L. monocytogenes.While microbiome-induced immune homoeostasis is primarily associated with immune suppression, up-regulation of TNFα expression by probiotic administration has been shown to influence hepatic, as well as gastrointestinal, innate immune signalling. 11he impact of gut Akkermansia interactions upon the systemic immune system is the subject of intense study.Akkermansia outer membrane protein Amuc_1100 was shown to interact with enterocyte host Toll-like receptor (TLR) 2 and improve gut barrier function in mice. 8TLR2 is itself part of the TNFα signalling pathway in response to bacterial infection 12 and Akkermansia Amuc_1100 has been shown to activate nuclear factor kappa B in vitro. 13Improved barrier function following Akkermansia administration is linked to a reduction in systemic lipopolysaccharide (LPS) with broad consequences for systemic immune regulation and macrophage infiltration into adipocytes in the context of obesity. 8Recent studies have demonstrated that administration of Akkermansia or Amuc_1100 locally in the oral cavity improved bone loss in an experimental model of inflammatory Porphyromonas gingivalis-induced periodontitis and had systemic effects upon inflammation. 14Furthermore, a tripeptide derived from Akkermansia (Arg-Lys-His; RKH) can prevent lethal sepsis in a variety of model systems. 15Akkermansia has also been shown to specifically modulate the adaptive immune response influencing T cell and antibody responses in the host; factors that potentially influence cell-mediated responses to pathogens and pathobionts. 16Indeed, the systemic immune-modulatory effects of orally administered live or pasteurised A. muciniphila have been linked to reduced pulmonary viral load in a mouse model of influenza infection. 17Another detailed study showed that A. muciniphila produces the β-carboline alkaloid harmaline which protects against phlebovirus (a member of the Bunyaviridae) through systemic immune modulation via a mechanism that involves enhancement of liver bile acid production and subsequent immune-signalling via transmembrane G-protein coupled receptor-5 (Figure 1). 18kkermansia also displayed systemic effects in pathologies other than infection.It ameliorated harmful phenotypes associated with obesity by modulating host urinary metabolomic profiles and intestinal energy absorption. 8Akkermansia also appears to mitigate the negative effects of systemic interferon gamma on glucose metabolism, with significance for diabetes. 19Akkermansia has even been shown to ameliorate chronic stress-induced depressive symptoms in mice, implicating it in the gut-brain axis, 20 and recent evidence shows that gut motility and glycaemic control is influenced through Akkermansia triggering gut neurons to influence hypothalamic nitric oxide signalling. 21All of this evidence demonstrates that the physiological influence of Akkermansia goes beyond the local environment of the gut and can moderate systemic Akkermansia muciniphila mediates its effects on a local and systemic level.In the intestine, Akkermansia influences the number of goblet cells and down-regulated TNFα expression. 4Its outer membrane protein, Amuc_1100, was shown to activate TLR2 and improve barrier function with an associated reduction of systemic LPS (a key driver of systemic inflammation), 8 while it may also modulate immune homoeostasis by influencing IgA and local adaptive immune responses. 16A. muciniphila has the capacity to influence systemic immune parameters via a variety of proposed mechanisms. A. muciniphila produces the β-carboline alkaloid harmaline which protects against phlebovirus infection in the lungs through a proposed mechanism that involves up-regulation of bile acid synthesis in the liver and modulation of immune signalling via the TGR5 bile acid receptor. 18The tripeptide RKH is produced by Akkermansia and has been shown to reduce TLR4 signalling pathways resulting in moderation of potentially lethal sepsis. 15inally, A. muciniphila is known to activate the enteric nervous system and impact signalling to the hypothalamus, 21 although the implications of this interaction for immune homoeostasis are currently unclear.Figure was generated using Biorender.TGR: transmembrane G-protein coupled receptor; TLR: Toll-like receptor; TNFα: tumor necrosis factor-alpha.
immune-metabolic-inflammatory pathways with implications for host health.Some of these potential mechanisms are outlined in Figure 1.
Whilst we examined Akkermansia administration and Listeria infection in the context of an HF diet, we have not yet determined the potential impact in mice fed normal chow or a low-fat diet.There remains the possibility that the effects are diet-dependent and result from amelioration of the detrimental effects of HF diet by Akkermansia administration in our model.Furthermore, potentially harmful effects of Akkermansia overabundance have been noted and discussed previously. 22Mucous degradation by Akkermansia in the gut stimulates enhanced mucous secretion benefiting host barrier function and impacting glucose metabolism and adiposity (reviewed in Chiantera et al. 22 ).However, mucous degradation may potentially damage the gut barrier, and Akkermansia administration has been associated with enhanced inflammation in mouse models of inflammatory bowel disease and enhanced tumorigenesis in a model of colon cancer. 22In a reduced complexity microbiota model (the SHIUMI model), the addition of Akkermansia actually promoted Salmonella infection and exacerbated inflammatory scores. 23However, the effects may be dependent on microbiota complexity as administration of both live Akkermansia or a pasteurised preparation reduced burden of Salmonella infection in a conventionally raised murine infection model. 24n summary, studies have demonstrated a capacity for Akkermansia to reduce infectious load in L. monocytogenes, C. difficile, Salmonella and virus-infection models through influences upon gut barrier function and the local and systemic immune system. 4,10,17,18,24However, further studies are required to investigate mechanisms in more detail, to further examine the systemic immune-modulatory effects, and to determine impacts against other infectious agents.It is notable that the safety and clinical efficacy of live and pasteurised A. muciniphila in reducing several parameters of metabolic syndrome in humans was demonstrated as part of clinical trial NCT02637115. 22urthermore, pasteurised A. muciniphila has been classified as a novel food by the European Food Safety Authority, permitting its sale and consumption by the public outside of a clinical setting. 22This establishes A. muciniphila as a next-generation probiotic, supplementing the established genera of Lactobacillus and Bifidobacterium.Our work demonstrates potential impacts of Akkermansia upon infection and adds to the growing armoury of commensal strains that may have the potential to limit infectious disease.Together with the work of others, this suggests a novel approach to the prevention and limitation of infectious disease through understanding and implementing alterations to the microbiota. 3,25,26

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/ or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/ or publication of this article: This work was supported by Science Foundation Ireland (grant numbers 16/IA/4445 and SFI/12/RC/2273_P2).The authors acknowledge funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 956279.

Figure 1 .
Figure1.Akkermansia muciniphila mediates its effects on a local and systemic level.In the intestine, Akkermansia influences the number of goblet cells and down-regulated TNFα expression.4Its outer membrane protein, Amuc_1100, was shown to activate TLR2 and improve barrier function with an associated reduction of systemic LPS (a key driver of systemic inflammation),8 while it may also modulate immune homoeostasis by influencing IgA and local adaptive immune responses.16A. muciniphila has the capacity to influence systemic immune parameters via a variety of proposed mechanisms. A. muciniphila produces the β-carboline alkaloid harmaline which protects against phlebovirus infection in the lungs through a proposed mechanism that involves up-regulation of bile acid synthesis in the liver and modulation of immune signalling via the TGR5 bile acid receptor.18The tripeptide RKH is produced by Akkermansia and has been shown to reduce TLR4 signalling pathways resulting in moderation of potentially lethal sepsis.15Finally, A. muciniphila is known to activate the enteric nervous system and impact signalling to the hypothalamus,21 although the implications of this interaction for immune homoeostasis are currently unclear.Figure was generated using Biorender.TGR: transmembrane G-protein coupled receptor; TLR: Toll-like receptor; TNFα: tumor necrosis factor-alpha.